Glutathione HiCap represents a state-of-the-art matrix for affinity purification of GST and GST-fusion protein. Purification of these GST-tagged proteins is achieved by their strong and specific binding to the tripeptide ligand glutathione, which is stably immobilized on the bead support of the matrix. Glutathione HiCap Matrix provides high binding capacity for GST-tagged proteins and ensures high-resolution affinity purification of GST and GST-fusion proteins. The Glutathione HiCap Matrix is available in both bulk and cartridge formats.

Purification of GST-tagged protein was performed with Glutathione HiCap Matrix and resins from various other suppliers. High purity was achieved using QIAGEN's new Glutathione HiCap Matrix. M: Marker; CL: Cleared lysate. Elution fractions are shown. Analysis was performed by SDS-PAGE. *Glutathione resin; †Fast-flow resin; ‡High-performance resin

Glutathione HiCap resin represents a state-of-the-art matrix for affinity purification of GST and GST-fusion proteins in a single-step process (see figure"Glutathione affinity purification"). Using a simple bind-wash-elute procedure, GST-tagged protein from various sources, both native and recombinantly expressed, are strongly and specifically bound to the tripeptide ligand glutathione, which is stably immobilized on the matrix bead support. Non-tagged proteins are washed through and GST-tagged proteins are easily obtained with glutathione in the elution buffer, giving high yields of tagged proteins (see figures "Purification of GST-tagged protein", and "Delivery of highly pure proteins").

The Glutathione HiCap Matrix is an excellent choice for column purification and scale-up (cartridges or custom-packed columns). Glutathione HiCap Cartridges are pre-filled with 1 ml or 5 ml Glutathione HiCap Matrix and are ready to use for purification of GST-tagged proteins using a syringe, peristaltic pump, or automated liquid chromatography systems (such as ÄKTA, ÄKTAdesign, or BioLogic workstation). The highly physically stable bead support (WorkBeads) is made from agarose, providing reliable purification under a variety of conditions. Excellent flow characteristics of the HiCap Matrix make it highly suited for automated systems, and provide high dynamic binding capacity. Glutathione HiCap Matrix is suitable for batch purification, gravity-assisted chromatography, and packed-bed column chromatography (cartridges or custom-packed columns), as well as for high-throughput applications in vacuum- or centrifugation-based protocols. The outstanding stability of the matrix also means that Glutathione HiCap Matrix is well suited for GST pull-down applications to determine the physical interaction between proteins, where the beads are subjected to repeated high g-force centrifugation steps.

Procedure

In the single-step purification of GST-tagged proteins (see figure "Glutathione affinity purification"), lysate containing the tagged protein is loaded onto the column/cartridge. After initial washing with column buffer, the GST-tagged protein is collected using elution buffer containing reduced glutathione, which competes with the matrix-bound reduced glutathione for the GST-tagged protein.